The present disclosure relates generally to the field of nucleic acid sequencing techniques. More particularly, the disclosure relates to techniques for enriching target capture and reducing off-target capture of nucleic acids to be sequenced in a targeted sequencing workflow.
Sequencing methodology of next-generation sequencing (NGS) platforms typically makes use of nucleic acid fragment libraries. In targeted sequencing techniques, a subset of fragments containing genes or regions of interest of the genome are isolated from the nucleic acid library and sequenced. Targeted approaches using NGS allow researchers to focus time, expenses, and data analysis on specific areas of interest. Such targeted analysis can include the exome (the protein-coding portion of the genome), specific genes of interest (custom content), targets within genes, or mitochondrial DNA. Targeted approaches contrast with whole genome sequencing approaches that are more comprehensive, but that also involve sequencing regions of the genome that may not be of interest to all users.
In one example of a targeted sequencing technique, hybrid capture methods use a panel or set of probes that hybridize to target sequences in the nucleic acid library. Hybridization of the probes to the target sequences allows these sequences to be separated from the rest of the fragments in the library for sequencing. By targeting only a portion of the nucleic acid library, hybrid capture methods avoid sequencing of off-target nucleic acid fragments that do not contain sequences of interest. However, unlike amplicon-based target enrichment methods, hybrid capture methods have a higher rate of off-target sequencing and, in turn, lower on-target specificity. For example, certain hybrid capture methods generally achieve only 40%˜60% efficiency, despite the use of commercial hybridization blockers such as Cot1, tRNA, salmon sperm DNA, poly(dIdC) and blockers targeting the universal adapters of library fragments. The off-target reads not only waste sequencing yield, but also potentially compromise variant calling for somatic mutations of low frequency. Therefore, there is a need for improved enrichment methods that provide for higher specificity in targeted sequencing techniques.